Genes involved in the molecular paths of tumor suppression and/or resistance to viruses

ABSTRACT

The invention concerns genes involved in the molecular paths for tumor suppression and/or resistance to viruses, and whereof the cell expression is in particular induced or inhibited during apoptosis and/or tumor suppression.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national stage application, filed under 35U.S.C. § 371, and claims foreign priority to French patent applicationNo. 98/10077, filed Aug. 5, 1998.

BACKGROUND OF THE INVENTION

The present invention relates to the revelation of genes involved in themolecular pathways of tumor suppression and/or resistance to viruses.

1. Field of the Invention

The present invention has been made possible by the isolation of cDNAcorresponding to messenger RNAs expressed or repressed during tumorsuppression and/or during the process of apoptosis induced by the p53suppressor gene.

2. Description of the Art

One of the most important suppressor genes involved in apoptosis is thep53 gene. When functioning normally, this gene controls cell growth andthe process of apoptosis; in particular, it is this gene which blockscell growth and which must induce the apoptotic process in order toavoid the development of a cancer. It has thus been demonstrated thatmice nullizygous for p53 are much more sensitive to the formation oftumors. The fact that, in cancers, the p53 gene is very often modifiedand leads to the production of proteins incapable of vehicling themessage of apoptosis has also been demonstrated.

It is this particularity which has been used in the context of thepresent invention.

Specifically, the present invention is based on the observation that itis not possible, or at least that it appears to be very difficult, toset up a direct substitution therapy when there is dysfunctioning of thep53 gene. Specifically, p53 which is mutated, as it is in cancer, willannul the physiological effect of normal p53.

It has therefore been necessary to abandon, at least initially, the ideaof a substitution therapy acting directly on p53.

The present invention has, therefore, endeavored to study the geneslocated upstream and downstream of p53 in order to bypass the difficultymentioned above.

In order to isolate the genes activated or inhibited by normal p53(wild-type p53), an overall combing of gene expression has been carriedout in a malignant line (K562) and a derived cell (KS) in which themalignant phenotype is suppressed, more particularly in a cellexpressing p53 which is functionally normal (KS) and in a cell notexpressing p53 (K562). Comparison of the genes expressed (messenger RNAsexpressed in the two types of cell) has made it possible to reveal genesexpressed differentially, i.e. expressed in one of the cells whereasthey are not expressed in the other (the genes may be activated orinhibited).

It is easily deduced therefrom that these genes are involved in thecancerization process, in one case by their absence and, in the othercase, by their presence.

The method used for this differential study is the method described in1992 by Liang and Pardee (Differential display of eukaryotic mRNA bymeans of a polymerase chain reaction).

SUMMARY OF THE INVENTION

The approach to the problem according to the present invention has madeit possible to isolate sequences directly linked to a function.Consequently, unlike the random sequencing of ESTs, the sequences aresequences the function of which is known and which are involved in theprocess of suppression of the malignant phenotype and/or of apoptosisinduced by the p53 suppressor gene, and/or in resistance to viruses.

Thus, the present invention relates to novel sequences and the genescomprising them, and to the use of these sequences, both at thediagnostic and at the therapeutic level, just as for producing modelsintended to test anticancer and antiviral products.

The present invention relates, first of all, to a nucleotide sequencecorresponding to a gene comprising:

-   (a) a sequence according to one of the SEQ IDs 1 to 15 or an    equivalent gene which comprises:-   (b) a sequence which hybridizes with one of the sequences according    to (a),-   (c) a sequence which has at least 80% homology with (a) or (b), or-   (d) a sequence which encodes a protein encoded by a gene according    to (a), (b) or (c), or an equivalent protein, and to their use in    particular in cancer suppression and/or resistance to viruses and in    therapeutic monitoring.

It should be recalled that sequences 1 to 15 constitute only a part ofthe genes implicated, but that the present invention covers both thenucleotide sequence corresponding to the whole gene and fragments of hisgene, in particular when they encode an equivalent protein as will bedescribed hereinafter.

The nucleotide sequences can be both DNA sequences and RNA sequences, orsequences in which some of the nucleotides are unnatural, either inorder to improve their pharmacological properties or in order to enabletheir identification.

The sequences mentioned in (b) are essentially the entire or partialcomplementary sequences (in particular for the cases mentioned above)

Thus, the invention also relates to the nucleotide sequences of thegenes which have strong homology with the genes mentioned above,preferably greater than 80% homology on the essential portions of saidgenes, namely in general at least 50% of the sequence, preferably thehomology will be greater than 90% on these portions.

Finally, when said genes encode a protein, the present invention alsorelates to the sequences encoding the same protein, taking into accountthe degeneracy of the genetic code, but also the equivalent proteins,i.e. the proteins producing the same effects, in particular the deletedproteins and/or the proteins which have undergone point mutations.

DETAILED DESCRIPTION OF THE INVENTION

The sequences according to the present invention are more particularlythe sequences which are induced or inhibited during cellular apoptosis,in particular those induced by p53 and/or p21 and/or TSAP 3 (HUMSIAH)and/or antisense-TSIP 2 (antisense-PS1). In other words, these sequencescorrespond to genes the cellular expression of which is activated by oneat least of the transfectants chosen from the group comprising the p21transfectants, the TSAP 3 transfectants and the antisense TSIP 2transfectants.

Said genes are grouped together as TSAP or “Tumor Suppressor ActivatedPathway” and termed from TSAP 9 to TSAP 22, corresponding to SEQ IDs 1to 14, and as TSIP or “Tumor Suppressor Inhibited Pathay”, and termedTSIP 3, corresponding to SEQ ID 15.

The characteristics of the sequences are given in the tables appendedherein.

The nucleotide sequences corresponding to the TSAP genes are sequenceswhich are expressed during the process of apoptosis, whereas when theyare not expressed, the process of oncogenesis continues. It is thereforeadvantageous:

-   -   to detect any abnormality in the corresponding gene, which        abnormality may lead to greater susceptibility to oncogenesis,        and    -   to be able to provide a replacement therapy.

It should, moreover, be recalled that these genes may be involved inprocesses other than the processes of tumor suppression; specifically,p53 is in some ways the guardian of the integrity of the genome andunder these conditions the TSAP or TSIP genes are doubtless alsoinvolved in this control function. It is, therefore, all of the possiblemodifications of the genome which may be liable to the detection and tothe therapy above. On the other hand, the TSIP genes are expressedduring oncogenesis and this expression is decreased, or even inhibited,during apoptosis and tumor suppression; it is therefore, in this case aswell, advantageous to detect the possible abnormality of the TSIPs andalso to provide an inhibition/blocking therapy.

The replacement therapy may be carried out by gene therapy, i.e. byintroducing the TSAP gene with the elements which enable its expressionin vivo. The principles of gene therapy are known. Specific viral ornonviral vectors can be used, for example adenoviruses, retroviruses,herpesviruses or poxviruses. Most commonly, these vectors are used indefective forms which will serve as vehicles for TSAP expression with orwithout integration. The vectors can also be synthetic, i.e. mimic viralsequences, or consist of naked DNA or RNA according to the techniquedeveloped in particular by the company VICAL.

In most cases, it will be necessary to provide targeting elements whichensure tissue- or organ-specific expression; specifically, theactivation of an uncontrolled phenomenon of apoptosis cannot beenvisaged.

The present invention relates, therefore, to all of the vectorsdescribed above.

The present invention also relates to the cells transformed with anexpression vector as described above, and to the protein which can beobtained by culturing transformed cells.

The expression systems for producing proteins can be both eukaryoticsystems, such as the vectors above, and prokaryotic systems in bacterialcells.

I. [sic] one of the advantages of the present invention is that it hasdemonstrated the involvement of several genes in apoptosis; thus, theoverexpression of one of the genes by gene therapy can, for some ofthem, drive to apoptosis only the cells in which other disturbed genesare already expressed, i.e. malignant cells.

The present invention also relates, as a medicine, to a compound whichensures the cellular expression of at least one of the nucleotidesequences above when it is induced during apoptosis and/or tumorsuppression, in particular TSAP 9 to TSAP 22 genes, or conversely, whichensures the inhibition of the cellular expression of at least onecellular sequence as described above when it is inhibited duringapoptosis and/or tumor suppression, in particular TSIP 3. It may, forexample, be an activated nucleotide which ensures the blocking of thenucleotide sequence or be a monoclonal antibody directed against theprotein(s) encoded by the nucleotide sequence.

Moreover, it is possible to envisage approaches other than gene therapy,in particular the use of nucleotide sequences in a sense or antisensestrategy, i.e. which can block the expression of TSIP or, on the otherhand, which act upstream, promoting the expression of TSAP.

It is also possible to envisage a direct replacement strategy byproviding proteins corresponding to TSAP, or inhibitory antibodiescorresponding to TSIP.

Finally, it is possible to envisage the use of nonprotein molecules, theactivity of which will be to activate TSAP or to mimic the action of itsexpression product, or to inhibit TSIP or to block the action of itsexpression product.

These products can be easily tested on the modified cells which aredescribed in the examples, by introducing the products to be tested intothe cell culture and detecting the appearance of the apoptoticphenomenon. In the DNA, RNA or protein strategies, the products are ofcourse developed as a function of the sequences which are described.

The present invention relates, in particular, to the use of themedicines above as an anticancer agent.

However, the product of the TSAP 9 to 22 and TSIP 3 genes is also usefulas an antiviral agent, as will become apparent upon reading the example.

The present invention also relates, therefore, to the use of themedicines above as an antiviral agent.

In addition, the present invention relates, as a diagnostic agent fordetermining the predisposition to cancer, to all or part of thesequences according to the invention to be used as a nucleotide probe oras an amplification primer, but also, as a diagnostic agent fordetermining the predisposition to cancer, to an anticen corresponding toall or part of the proteins encoded by the sequence according to theinvention, or the corresponding antibodies, optionally after culturing.

The diagnostic methods are known; they may be, for example, techniquesfor microsequencing the variable portions after isolation and optionalamplification, or methods for RFLP-detection or for simple amplificationin particular. Differential techniques can, in particular, make itpossible to demonstrate the difference between the normal and abnormalTSAP (or TSIP).

The invention also relates to the models using the sequences above.

Moreover, it should be emphasized that the inventors have demonstrated,by extension of the sequences in accordance with the invention initiallyrevealed, homologies shown by said extended sequences with sequencescorresponding to known proteins.

More particularly, besides the homology of TSAP 9 with a mousechaperonin containing the TCP-1 gene (9), the inventors have brought tolight a strong homology shown by TSAP 13 with the p40.5 subunit of thehuman proteasome (10, 11) and a strong homology shown by TSAP 21 withsyntaxin 11 belonging to the group of SNARE proteins (12).

The chaperonins are involved in the process of protein folding andassembly in the eukaryotic cytosol. They are suspected of slowing downthis folding by trapping intermediates which otherwise would aggregate.Among the proteins on which the chaperonin containing the TCP-1 genehomologous to TSAP 9 would act, mention may be made of actin, tubulinand the capsid protein of the hepatitis B virus.

The proteasome, in the same way as ubiquitin, is the main component ofthe major proteolytic system responsible for the degradation of manyintracellular proteins, including aberrant proteins resulting frommutations or from environmental stress. The p40.5 subunit of the human26S proteasome has recently been revealed, as well as its homolog inyeast Nas7p. In humans, the mRNA corresponding to the abovementionedsubunit is more particularly expressed in the pancreas, placenta,testicles, heart and skeletal muscle. It appears, moreover, that yeastcells deficient for Nas7p are particularly sensitive to heat stress.This contributes to the suggestion that the function of the 26Sproteasome is degraded during a heat stress.

The SNARE (Soluble N-ethylmaleimide-sensitive factor-attachment proteinreceptor) proteins are proteins the differential expression and theassociations of which are involved in the organization of the membranecompartments of cells. These proteins are specifically located in theregion of the Golgi apparatus, of the endosomes and of the lysosomes,which suggests that they play a role in the regulation of membraneexchanges using these organelles. More particularly, syntaxin 11 isthought to be located in the post-Golgi region.

It would be advantageous to be able to determine whether the molecularpathways in which the sequences in accordance with the invention areinvolved have common points with the molecular pathways in which theabovementioned proteins are involved, which would make it possible toenvisage novel modes of action on the abovementioned sequences for, forexample, therapeutic or diagnostic purposes.

Other characteristics of the invention will become apparent upon readingthe example below.

MATERIALS AND METHODS Cell Cultures

K562, KS, K52 and K53 cells were used as models. The K562 line is atumor line derived from a chronic leukemia of erythromyeloid type. It ischaracterized in particular by a Philadelphia chromosome which containsthe translocation (9,22) in which there is a rearrangement of the bcrgene with the abl proto-oncogene. This line has, moreover, an abnormalkaryotype and overexpresses the myc and pim-1 oncogenes. These lines aredescribed in the reference A. Telerman et al.: A model for tumorsuppression using H-1 parvovirus, Proc. Natl. Acad. Sci. USA. Vol. 90,pp. 8702-8706, September 1993.

In summary, a monoclone of K562 was infected with the H-1 parvovirus.This infection caused a massive death of the cell culture. Aftermaintaining this culture for a period of two months, the KS clone wasisolated. The same experiment carried out a second time provided, afterthree months of incubation, the KS2 and KS3 clones.

Using the same approach, the inventors derived, from a population ofU937 malignant cells, the US3 and US4 lines, which are resistant to theH-1 parvovirus and which show suppression of the malignant phenotype.These lines are described in reference (7).

M1 myeloid leukemia-cells and M1 cells were stably transfected with aheat-sensitive mutant val 135 p53 (LTR6).

These cells are cultured on RPMI 1640 medium with 10% FCS, at 5% of CO₂at 37° C. (3). For the temperature modification, the cultures are placedin a second incubator at 32° C.

U937 line transfected with p21^(WAF1): the complete coding portion ofthe cDNA of the p21^(WAF1) gene was cloned into the vector pBK-RSV(Stratagene, La Jolla, Calif.). 3.5 million U937 cells were transfectedwith 20 micrograms of DNA/30 micrograms of Lipofectin (LifeTechnologies).

The stable transfectants were selected using 1.5 mg/ml of G418 (Sigma).The characteristics of this Line portray in particular a suppression ofthe malignant phenotype.

U937 line transfected with TSIP 2 (PS1) in the antisense position: thecomplete coding portion of the cDNA of the TSIP 2 (PS1) gene was cloned,in the antisense position, into the vector pBK-RSV (Stratagene, LaJolla, Calif.). 3 million U937 cells were transfected with 20 microgramsof DNA/30 micrograms of Lipofectin (Life Technologies).

The stable transfectants were selected using 1.5 mg/ml of G418 (Sigma).The characteristics of this line, portraying in particular a slowingdown of growth, activation of apoptosis and suppression of the malignantphenotype, have been described in reference (8).

U937 line transfected with TSAP3 (HUMSIAH): the complete coding portionof the cDNA of the TSAP 3 gene was cloned into the vector pBK-RSV(Stratagene, La Jolla, Calif.). 3 million U937 cells were transfectedwith 20 micrograms of DNA/30 micrograms of Lipofectin (LifeTechnologies).

The stable transfectants were selected using 1.5 mg/ml of G418 (Sigma).The characteristics of this mine comprise in particular activation ofapoptosis and suppression of the malignant phenotype.

Study of the Differential cDNAs

In order to carry out the tests under standard experimental conditionsand to obtain total reproducibility of the results, the followingmodifications to the protocol of origin (1) were carried out:

PolyA+ mRNAs purified twice on an oligodT column using Fast Track(Invitrogen, San Diego Calif.) are always used. After reversetranscription (M-MLV Reverse Transcriptase, Gibco BRL) on 0.05 μg ofpolyA+ using 20 μM of each of the dNTPs (Boehringer-Mannheim), no addeddNTP is added to the final PCR mixture. A “hot start” at 94° C. for 5minutes is carried out before the PCR (GeneAmp PCR system 9600 PerkinElmer Cetus). The samples are cooled rapidly on iced water. A “touchdown” (2) of 10 cycles of 50° C. to 40° C. is carried out (94° C. 30seconds—50° C. 1 minute—72° C. 30 seconds), followed by 35 cycles (94°C. 30 seconds—40° C. 1 minute—72° C. 30 seconds, and a final extensionof 5 minutes at 72° C. The PCR products are separated on nondenaturing6% polyacrylamide gels (4). The gels are exposed without drying. Eachdifferential presentation is carried out by comparing M1S6 and LTR6 at37° C. and after incubating the two cell lines at 32° C for 4 hours.

The differential presentation procedure is repeated in 3 differentexperiments in order to confirm total reproducibility.

The differentially expressed bands are cut out of the gel, eluted andreamplified (1). The PCR products are subcloned using the TA-cloningsystem (Invitrogen, San Diego, Calif.), following the instructionsprovided.

For each ligation reaction, 10 recombinant clones are sequenced usingthe ABI automatic system.

RNA Extraction, Analyses and Northern Blot Probes

The total RNA is extracted with Trizol (Life Technologies). The poly1+[sic] RNAs are prepared using the OligotexdT kit (Qjagen, Calif.). 30 μgof the total RNA or 2 μg of polyA+ RNA are separated on 1% agarose,1×MOPS/2% formaldehyde gel and transferred onto nylon membrane (HybondN+, Appligène, France) as has been described previously (5). TheNorthern blots are hybridized with probes labeled with P³² on the TSAPand TSIP inserts, and washed as described previously (5). In order toverify the induction of the wild-type p53 function, the Northern blotsare hybridized with a cyclin G probe (6). By way of control for theamount of mRNA loaded, the blots are hybridized with a GAPDH probe.Various Northern blots (Clontech Calif.) are used under identicalconditions and hybridized for control with a β-actin probe. The Northernblots are exposed for 10 days at −80° C.

EXAMPLE 1

The desired aim is to characterize the molecular pathways which lead tothe suppression of cancer.

The following hypothesis was made to develop a model: if it was possibleto select, from a tumor which is sensitive to the cytopathic effect ofthe H-1 parvovirus, the cells which were resistant, this resistancemight be due to a change in their malignant phenotype. It was possibleto demonstrate this for the KS cells selected from the K562erythroleukemia cells. Unlike the parental K562 line, the KS, KS2 andKS3 clones are resistant to the cytopathic effect of the H-1 parvovirus.In addition, the tumorigenicity of the KS, KS2 and KS3 cells is reducedby 90%, while, when cultured in soft agar, the tumorigenicity of thesesame KS lines in vivo when injected into Scid-Scid immunosuppressed miceis reduced. At the molecular level, it could be noted that thissuppression of the malignant phenotype went hand in hand with areexpression of the p53 suppressor gene.

15 cDNAs expressed differentially between the K562 and KS cells wereisolated. TSAP 9 is homologous to the chaperoning.

Table 1 shows the molecules characterized, giving the primers and thesizes of the mRNAs detected by Northern blot.

Of these 15 molecules, all are induced in the KS cells, except TSIP 3,the expression of which is inhibited during the suppression of themalignant phenotype.

In transfection experiments, it was also possible to demonstrate thatthe resistance to the cytopathic effect of the H-1 parvovirus went handin hand with an intact function of the p53 gene and that cellstransfected with p53 mutants became sensitive to the cytopathic effectof the H-1 parvovirus.

The 15 molecules which we have isolated encode, therefore, genes whoseoverexpression (TSAP 9-TSAP 22) or inhibition (TSIP 3) is associated notonly with the suppression of cancer, but also with resistance to the H-1parvovirus. Consequently, these genes encode molecules which are part ofthe molecular pathways of cancer suppression and are potentialsuppressor genes.

In order to more clearly define the p53/p21 activation pathways, theinventors have studied:

-   -   the activation of these TSAPs/inhibition of the TSIPs in the        heat-sensitive p53 model developed in Moshe Oren,    -   the activation of these TSAPs/inhibition of the TSIPs in the        model in which U937 cells are transfected with the p21 gene,    -   the activation of the novel TSAPs/inhibition of the TSIPs in the        model in which U937 cells are transfected with the TSAP3 gene,        and    -   the activation of these novel TSAPs/TSIPs in the model in which        U937 cells are transfected with the TSIP 2 (PS1) gene in the        antisense position.

Table 1 below reports results of differential expressions analyzed byNorthern blot of the various probes (TSAP 9-TSAP 22, TSIP 3) of theK562/KS model and other U937/US3-US4 models, i.e. in a model of tumorsuppression in which the p21gene is activated via the p53 independentpathway. These cDNAs are therefore activated in two different cellularsystems of tumor suppression (the K562/K2 erythroleukemia model and theU937/US myelomonocytic model).

According to this table, it is noted that, in the majority of cases, thegenes expressed differentially in the K562/KS model are also expresseddifferentially in the U937/US3-US4 model. Molecular machinery for tumorsuppression therefore exists which is common to various types of cancer.This conclusion is also valid for the M1/LTR-6 model. It should benoted, in the latter case, that the absence of signals in certainTSAPs-TSIPs is probably due to the fact that the experiments werecarried out in two heterologous systems (human probes on mouse RNA).

TABLE 1 U937/US3-US4 CLONE WITH K562/KS MODEL M1/LTR-6 MODELDIFFERENTIAL 3′ AND 5′* K562/KS MODEL mRNA MRNA EXPRESSION PRIMERS cDNAPROBE HOMOLOGY mRNA kb RESULT kb RESULT kb TSAP 9 T11AA-9 K26 D3Chaperonin⋄ 2.6 DIFF. EXP. 2.0 DIFF. EXP. 2.6 TSAP 10 T11AA-9 K25.0 A111.6 DIFF. EXP. 1.6 NO SIGNAL 1.6 TSAP 11 T11AA-9 K25.0 B7 EST 2.9 NODIFF. 2.8 NO SIGNAL 2.9 EXP. TSAP 12 T11AA-9 K27.1 C7 EST 5.5 NO SIGNAL5.5 NO SIGNAL 5.5 TSAP 13 T11AA-23 K25.1 F3 Proteasome° 1.8 DIFF. EXP.1.5 DIFF. EXP. 1.8 TSAP 14 T11AC-5 K33.2 F10 EST 2.5 DIFF. EXP. 2.8DIFF. EXP. 2.5 TSAP 15 T11AG-19 K22 E3 EST 1.6 DIFF. EXP. 1.8 NO SIGNAL1.6 TSAP 16 T11GC-2 K12.1 F5 2.8 DIFF. EXP. 2.0 DIFF. EXP. 2.8 TSAP 17T11GC-12 K16.1 C7 1.8 DIFF. EXP. 1.9 DIFF. EXP. 1.8 TSAP 18 T11GG-5 K3.1D2 EST 2.0 NO DIFF. 1.8 DIFF. EXP. 2.0 EXP. TSAP 19 T11GG-23 K5.2 E10EST 1.5 DIFF. EXP. 1.6 NO SIGNAL 1.5 TSAP 20 T11GG-23 K5.1 A12 1.7 DIFF.EXP. 1.9 NO SIGNAL 1.7 TSAP 21 T11GG-23 K5.1 A1 SNARE^(Δ) 2.1 DIFF. EXP.1.9 DIFF. EXP. 2.1 TSAP 22 T11GG-5 K3.1 A12 EST 2.8 DIFF. EXP. 2.6 DIFF.EXP. 2.8 TSIP 3 T11AC-5 K33.1 B11 EST 9.5 DIFF. EXP. 9.5 NO SIGNAL 9.5*the numbers and sequences of the primers in the 5′ position correspondto those reported by Bauer et al. ⋄HUMKG1DD human mRNA for the ORF(human equivalent of mouse chaperonin containing the TCP-1 gene(t-complex polypeptide)). °p-40.5 subunit of the proteasome (Nas7p)^(Δ)SNARE syntaxin 11 DIFF. EXP. = Differential expression NO DIFF.EXPR. [sic] = No differential expression

Table 2 below summarizes the differential expression of certain TSAP andTSIP clones in various transfectant lines.

It emerges from this table that, in the majority of cases, the p21transfectants, TSAP 3 transfectants or antisense-TSIP2 transfectants arecapable of activating the molecular machinery of tumor suppressioncommon to the U937/US and K562/KS systems.

TABLE 2 ANTISENSE P21 TRANS- TSAP3 TRANS- TSIP2 TRANS- FECTANTS FECTANTSFECTANTS DIFFERENTIAL DIFFERENTIAL DIFFERENTIAL CLONE EXPRESSIONEXPRESSION EXPRESSION TSAP9 YES YES YES TSAP10 YES YES YES TSAP11 NO NONO TSAP12 NO NO NO TSAP13 YES YES YES TSAP14 YES YES YES TSAP15 YES YESYES TSAP16 NO YES NO TSAP17 YES NO NO TSAP18 NO YES YES TSAP19 NO NO NOTSAP20 YES NO YES TSAP21 YES YES YES TSAP22 YES YES YES TSIP3 YES YESYES

Table 3 below recapitulates the characteristics of differentialexpression of the cDNA clones by Northern blot.

TABLE 3 U937 cDNA MRNA K562/K U937 U937 SIAH/ clones kb HOMOLOGY [sic]U937/US p21 AS PS1 TSAP3 TSAP9 2.6 Chaperonin⋄ D D D D D TSAP10 1.6 ESTD D D D D TSAP11 2.8 EST D N N N N TSAP12 5.5 EST D N N N N TSAP13 1.8Proteasome° D D D D D TSAP14 2.5 EST D D D D D TSAP15 1.6 EST D D D D DTSAP16 2.5 NO D D N N D TSAP17 1.8 NO D D D N N TSAP18 2.0 EST D N N D DTSAP19 1.5 EST D D N N N TSAP20 1.7 NO D D D D N TSAP21 2.1 SNARE^(Δ) DD D D D TSAP22 2.6 EST D D D D D TSIP3 9.5 EST D D D D D D: Differentialexpression N: No differential expression ⋄ Chaperonin containing the TCP1 gene ° p40.5 subunit of the proteasome (Nas 7p) ^(Δ) SNARE syntaxin 11

References

-   (1) Liang P. & Pardee A. B. (1992) Science, 257, 967-971-   (2) Don R. H., Cox P. T., Wainwright B. J., Baker K. &    Mattick J. S. (1991) Nucl. Acids Res., 19, 4008-   (3) Yonish-Rouach E., Resnitzky D., Lotem J., Sachs L., Kimchi A. &    Oren M. (1991) Nature 352, 345-347-   (4) Bauer D., Muller H., Reich J., Riedel H., Ahrenkiel V.,    Warthoe P. & Strauss M. (1993) Nucl. Acids Res. 21, 4272-4280-   (5) Sambrook J., Fritsch E. F. & Maniatis T. (1989) Molecular    Cloning: a laboratory manual-   (6) Okamoto K. & Beach D. (1994) EMBO J., 13, 4816-4822-   (7) Nemani M., Linares-Cruz G., Bruzzoni-Giovanelli H., Roperch    J.-P., Tuynder M., Bougueleret L., Cherif D., Medhioub M., Pasturaud    P., Alvaro V., Der Sarkissan H., Cazes L., Le Paslier D., Le Gall    I., Israeli D., Dausset J., Sigaux F., Chumakov I., Oren M., Calvo    F., Amson R. B., Cohen D. and Telerman A., Activation of the human    homologue of the Drosophila sina gene in apoptosis and tumor    suppression, Proc. Nati., Acad. Sci. USA (1996) 93, 9039-9057-   (8) Roperch J.-P., Alvaro V., Prieur S., Tuynder M., Nemani M.,    Lethrosne F., Piouffre L., Gendron M-G. , Israeli D., Dausset J.,    Oren M., Anson R., and Teleman A., Inhibition of presenilin 1    expression is promoted by p53 and p21 WAF-1 and results in apoptosis    and tumor suppression, Nature Medicine (1998) 4, 835-838.-   (9) Kubota et al., 1995, Eur. J. Biochem. 230, 3-16,-   (10) Hori et al., 1998, Gene, 216, 113-122,-   (11) Baumeister et al., 1998, Cell, Vol. 92, 367-380,-   (12) Advani et al., 1998, The Journal of Biological Chemistry, Vol.    273, No. 17, 10317-10324.

1. An isolated DNA molecule encoding TSAP 21, said isolated DNA moleculeconsisting of the nucleotide sequence of SEQ ID) NO:13, wherein theexpression of said TSAP 21 is activated by p53- or p21-induced apoptosisor tumor suppression.
 2. A vector comprising said isolated DNA moleculeof claim
 1. 3. The vector of claim 2, wherein said vector is a viralvector.
 4. The vector of claim 3, wherein said viral vector isadenoviral, retroviral, herpesviral or poxviral.
 5. The vector of claim2, wherein said vector is a plasmid.
 6. An isolated or cultured hostcell stably transformed with the vector of claim 2.